Neonatal Inhibition of DNA Methylation Disrupts Testosterone-Dependent Masculinization of Neurochemical Phenotype

Abstract

Many neural sex differences are differences in the number of neurons of a particular phenotype. For example, male rodents have more calbindin-expressing neurons in the medial preoptic area (mPOA) and bed nucleus of the stria terminalis (BNST), and females have more neurons expressing estrogen receptor alpha (ERα) and kisspeptin in the ventromedial nucleus of the hypothalamus (VMH) and the anteroventral periventricular nucleus (AVPV), respectively. These sex differences depend on neonatal exposure to testosterone, but the underlying molecular mechanisms are unknown. DNA methylation is important for cell phenotype differentiation throughout the developing organism. We hypothesized that testosterone causes sex differences in neurochemical phenotype via changes in DNA methylation, and tested this by inhibiting DNA methylation neonatally in male and female mice, and in females given a masculinizing dose of testosterone. Neonatal testosterone treatment masculinized calbindin, ERα and kisspeptin cell number of females at weaning. Inhibiting DNA methylation with zebularine increased calbindin cell number only in control females, thus eliminating sex differences in calbindin in the mPOA and BNST. Zebularine also reduced the sex difference in ERα cell number in the VMH, in this case by increasing ERα neuron number in males and testosterone-treated females. In contrast, the neonatal inhibition of DNA methylation had no effect on kisspeptin cell number. We conclude that testosterone normally increases the number of calbindin cells and reduces ERα cells in males through orchestrated changes in DNA methylation, contributing to, or causing, the sex differences in both cell types.

Disorders of Sex Development: Classification, Review, and Impact on Fertility

“…Indeed, hormonal influences not only affect internal and external genital development and differentiation; the embryo's brain has also been shown to differentiate sexually [12][13][14], perhaps through control mechanisms similar to those developed by the external genitalia [15]. On the other hand, it is also possible that the induction of the central nervous system (CNS) by hormones affects the patterns of hormonal secretion and, consequently, sexual behavior in adults [16][17][18][19][20]. Thus, the sex of an individual and their sexual expression, i.e., homoor heterosexual, must be considered the result of all influences that the individual receives, both before and after birth [8].…”

Sexually Dimorphic Formation of the Preoptic Area and the Bed Nucleus of the Stria Terminalis by Neuroestrogens

“…Compared with postnatal males, postnatal female rats have higher DNA methyltransferase activity in the POA, which is followed by higher DNA methylation, and postnatal estradiol treatment reduced DNA methyltransferase activity and DNA methylation in the female POA (Nugent et al, 2015). Moreover, inhibition of DNA methyltransferase in the brain of neonatal females increased dendritic spines of POA neurons and masculinized sexual behavior (Nugent et al, 2015), and increased the number of Calb neurons in the CALB-SDN and CALB-BNSTp, resulting in elimination of the sex difference in Calb neurons (Mosley et al, 2017;Cisternas et al, 2020). Epigenetic regulation via histone modification also contributes to masculinization of the brain.…”

DNA methylation and demethylation underlie the sex difference in estrogen receptor alpha in the arcuate nucleus

“…Epigenetic modifications, such as DNA methylation, play prominent roles in the differentiation of cell type throughout the body and may underlie sexual differentiation of ERα neurons [12][13][14][15][16]. For example, we recently found that inhibiting DNA methylation in newborn mice reduces or eliminates the usual female bias in the number of neurons expressing ERα in the preoptic area and ventrolateral area of the ventromedial nucleus of the hypothalamus (VMHvl), which lies adjacent to the ARC [14,16].…”